Cas9 mRNA containing 5-methylcytidines and pseudouridines was purchased from TriLink Biotechnologies. Cas9 protein was purchased from IDT. Cas9 protein and sgRNAs were complexed by incubation at a molar ratio of 1:2.5 at 25°C for 10 min immediately prior to electroporation. CD34+ HSPCs were electroporated 2 days after isolation, and T cells were electroporated 3 days after stimulation. All electroporations were performed on the Lonza Nucleofector 2b (program U-014 for HSPCs and T cells; program T-016 for K562 cells). For CD34+ HSPCs and T cells, either the buffer from the Human T Cell Nucleofection Kit (VPA-1002, Lonza) or the 1M electroporation buffer described in Chicaybam et al. (2013) was used. The following conditions were used for electroporation: 5–10 3 106 cells/mL, 300 mg/mL Cas9 protein complexed with sgRNA at a 1:2.5 molar ratio. For K562 electroporations, an electroporation buffer containing 100 mM KH2PO4, 15 mM NaHCO3, 12 mM MgCl2 3 6H2O, 8 mM ATP, and 2 mM glucose (pH 7.4) was used with 50 mg/mL Cas9 mRNA and 50 mg/mL sgRNA. For experiments with plasmid donors, 2.5 mg of each plasmid was used. For experiments using AAV6 donors, directly following electroporation, cells were incubated for 15 min at 37°C, after which, they were added AAV6 at 20% of the final culture volume (MOI was typically
2–5 3105 vector genomes (vg) per cell per AAV donor, unless otherwise stated).